The structure, function, and subunit interaction in the Alpha2Beta2 multienzyme complex of tryptophan synthase are being investigated. Since the Alpha subunit can be cleaved by limited proteolysis to yield an Alpha' derivative composed of 2 fragments, Alpha-1 and Alpha-2, which can be separated and shown to refold independently, these proteolytic fragments correspond to independent domains. The relationship between the domain structure of the Alpha subunit and the mechanism of guanidine hydrochloride-induced unfolding of the Alpha subunit has been investigated using circular dichroism and chemical modification studies. A partially unfolded intermediate has been characterized and shown to have the domain corresponding to the Alpha-2 fragment unfolded and the domain corresponding to the Alpha-1 fragment folded. The arrangement of the subunits, domains, and active sites in the Alpha2Beta2 complex is being investigated by chemical modification and crosslinking experiments. The stereochemistry of sodium borohydride reduction of Schiff bases of the coenzyme, pyridoxal phosphate, with substrates at the active site of the Beta2 subunit and with an active site lysyl residue, is being determined and compared.